Ligand for the neu gene product

ABSTRACT

A purified proteinaceous substance bindable with p185, the translation product of the neu oncogene is disclosed. The purified proteinaceous substance may be characterized in that it increases the activity of the tyrosine kinase contained in the neu oncogene product but does not increase the activity of tyrosine kinase of epidermal growth factor receptor; induces p185 dimerization and internalization; affects the growth of cells which express p185 in a dose dependent manner; is heat stable from about 56° C. to about 100° C.; is degradable by protease; and has a molecular weight of from about 7,000 to about 14,000 daltons in its smallest active form as determined by gel filtration and ultrafiltration membrane analysis. Methods of detecting p185 on the surfaces of tumor cells are also disclosed.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 505,837filed on Apr. 6, 1990, now abandoned, which is assigned to the assigneeof this application and is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of substances useful formodulating the metabolism of cells; more particularly the presentinvention relates to the field of proteinaceous substances useful formodulating the metabolism of cells.

BACKGROUND OF THE INVENTION

The neu gene product is a transmembrane growth-factor-receptor-liketyrosine kinase. It was originally isolated from chemically induced ratneuroblastomas that developed in the offspring of rodents exposed toethylnitrosourea at a discrete time period of gestation. The chemicallyinduced mutagenic event results in a point mutation (an adenine tothymidine) in the neu gene at the nucleotide level which translates intoa single amino acid substitution (valine to glutamic acid) in the neugene product's transmembrane region. The tyrosine kinase domain of therat neu gene product becomes constitutively activated by this pointmutation in its transmembrane region. The neu gene encodes a 185 Kdsurface glycoprotein, termed p185, that possesses tyrosine kinaseactivity and is structurally similar to the epidermal growth factorreceptor (EGFR) at the nucleotide and amino acid level. However, the neugene has been shown to be distinct from the epidermal growth factorreceptor-encoding gene (the c-erb-B gene) by detailed molecular analysisand chromosomal localization studies. The neu gene product's similaritywith the epidermal growth factor receptor (EGFR) suggests that p185 isalso a growth factor receptor of an as yet unidentified growthmodulating factor. The rat and human neu genes are 90% homologous. Therelative molecular masses of their protein products differ slightly,Mr=185 kDa for the rat protein and 190 kDa for the human protein. Thisdiscrepancy is thought to result from interspecies differences inpost-translational modification. See Greene, M. I. et al., "ReceptorSystems in Tissues of the Nervous System", Immunological Reviews, Number100, December 1987 for a review of the neu oncogene, its product andfunction.

The neu gene and neu gene product refers herein to all mammalian andvertebrate homologies of this gene and its protein product. As usedherein, the oncogenic form of the rat neu gene product will be denotedas p185neu. The normal cellular non-oncogenic gene product will bedenoted as p185c-neu. The human homologue of the rat neu gene product isreferred to herein as c-erb-B-2 or human neu. p185 written alone broadlyrefers herein to rat and human and other mammalian homologues of the ratneu gene product. p185 involvement in neoplasia and its growth factorreceptor like attributes suggest that p185 protein plays an importantrole in normal and abnormal growth and differentiation of the cells inwhich it is expressed.

p185 has been found in variety of tissues derived from developing andadult animals in a developmental stage and tissue specific manner,Kokai, Y. et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84: 8498-8501;Maguire, H. C. et al., (1989) J. Investigative Dermatology 92: 786-790;Cohen, J. A. et al, (1989) Oncogene 4: 81-88. Expression of p185 in theadult rat and human has been detected in the epithelial layers ofintestinal villi, basal layer and hair follicles of the skin, pulmonarybronchioles, proximal renal tubules, fallopian tubes, mammary ducts,bladder, and uterine endometrium and in some developing and adultperipheral and central glial cells. Neural and connective tissuesexpress neu during a relatively narrow time window in mid to lategestation, but show no expression in the adult. In secretory epitheliallayers of other organs, expression of the neu gene persists intoadulthood. Lymphoid tissues do not appear to express the neu gene at anydevelopmental stage.

p185c-neu is expressed on the surface of a number of normal cell typesand on the surface of some tumors. Minimal expression of p185c-neu hasbeen found in ependyma, choroid plexus, ciliary body, terminal bronchialepithelium, ovarian stromal epithelium and the loop of Henle.Genitourinary epithelium and normal skin appendages had slightly higherexpression. These tissues include bladder transitional epithelium,fallopian tube epithelium, bile duct, collecting duct, endometrial glandepithelium, epidermis, hair follicles, sebaceous gland, and uretheralepithelium. Higher expression of p185 has been found in the rapidlydividing tissues of breast and gastrointestinal tissue. These structuresinclude breast alveolar and ductal epithelium, hepatocytes, proximal anddistal tubules, pancreatic islet cells, and gastric mucosa. The highestlevels of p185 expression have been found in the rapidly dividingtissues of secretory epithelium and include the meibomian gland of theeye, the cornea, intestinal villus epithelium, pancreatic acinus,pancreatic ductal epithelium, and salivary ductal epithelium.Differential expression of p185 has been found in several types oftissues. In the kidney, the proximal and distal tubules have highexpression, whereas there is diminished expression in the Loop of Henle.In the small bowel epithelium, there is minimal expression in thecrypts, with gradually increasing expression as the villus tip isreached. In skin, there is minimal expression in the basal layers andincreasing expression in the epidermis. There is also staining in thehair follicles.

In human and rat tissues, static tissues and tissues with a slow rate ofadult cellular turnover do not express p185. These tissues are ofendodermal and mesodermal origin and included lymphoid tissue. Tissueswith no expression of p185 include adrenal, blood vessel, brainparenchyma, cartilage, epididymis, heart, lymph node, spleen, striatedmuscle, testis, thymus, and thyroid.

Although the neu gene has been cloned, identification of the primaryligand for its protein product has been difficult. Though severalendogenous p185 modulatory factors may exist, the effect of the primaryligand on p185 should be similar to those of EGF and PDGF on theirreceptors since these receptors are tyrosine kinases closely related top185. Yarden, Y., (1988) Annual Review of Biochemistry 57:443; Yardenand Schlessinger,(1987) Biochem. 26:1443; Bishayee, et al., (1989) J.Biol. Chem. 264:11699.

It is an object of the invention to provide substances and methods foraltering the cellular metabolism of mammals, particularly humans. It isalso an object of the invention to provide substances for treatingmammalian tumors, particularly human tumors. Current tumor treatmentsrely for the most part in the cytotoxic effects of drugs andradiological therapy. Although these treatments bring remission and cureto some patients, they unfortunately have serious side effects becausethey kill not only tumor cells but also some normal non-tumorous cells.There exists a great need for mammalian tumor treatments which affectprimarily the tumor cells, but that have minimal interference withnormal cells and cellular functions. It is a further object of theinvention to provide methods for diagnosing tumors expressing p185 onthe surfaces of the cells. Amplification of human neu gene andsubsequent overexpression of the human neu gene product has beenimplicated in adenocarcinomas in several tissue types including breast,stomach, colorectal, ovary and pancreatic tissue. As a result, c-erbB-2protein expression levels appear to be a useful prognostic indicator ofbreast, ovarian and lung cancers. These and other objects will becomeapparent to persons of ordinary skill in the art from a review of thepresent specification and the appended claims.

SUMMARY OF THE INVENTION

The present invention provides a purified proteinaceous substance whichis selectively bindable with p185, the translation product of the neugene and a method for purifying the proteinaceous substance. Theproteinaceous substance of this invention has been named NAF whichstands for "neu protein activating factor". When referenced herein,"NAF", "neu protein activating factor" and "proteinaceous substance" areequivalent and refer to the same compound. NAF increases the activity ofthe tyrosine kinase contained in the p185, but does not increase theactivity of tyrosine kinase of the epidermal growth factor receptor. Theproteinaceous substance induces p185 dimerization and internalization.In addition the proteinaceous substance of the invention affects theproliferation of neu-bearing cells. NAF is heat stable at 56° C. and 100° C. and is degradable by some proteases. The molecular weight of itssmallest active/isolatable form is between about 7,000 and about 14,000daltons. Forms of NAF having a molecular weight up to about 30,000daltons exist with a second active range identified as occuring betweenabout 14,000 and about 24,000 daltons. These larger forms may representoligomeric forms of the smallest active/isolatable form.

In other embodiments, the invention also provides a method of treating amammal to alter the metabolic state of the mammal which comprisesadministering the proteinaceous substance or a fragment thereof to themammal in an amount effective to alter the metabolic state of themammal. The invention additionally provides methods of treatingmammalian tumors expressing p185. The proteinaceous substance andmethods of the invention are useful for treating any type of cells whichexpress p185 normally, as altered forms, or at altered levels.

The invention is more particularly pointed out in the appended claimsand is described in its preferred embodiments in the followingdescription.

DETAILED DESCRIPTION OF THE INVENTION

NAF may be purified from medium conditioned by T cells which have beentransformed by human T cell lymphotrophic virus-1 (HTLV-1). A preferredcell line is ATL-2, an interleukin-2-independent HTLV(+) T cell line.The ATL-2 cell line was deposited in the American Type CultureCollection, Rockville, Md. on Mar. 23, 1990 and has accession number CRL10388. The endogenous source of this factor has proven to be elusive,perhaps because quantities of the factor are too low to be detected bythe assays described herein.

In a preferred embodiment of the invention, ATL-2 cells were washed twotimes with PBS, then cultured at 3×10⁵ /ml in serum-free RPMI 1640medium (Whittaker M. A. Bioproducts, Inc., Walkersville, Md.)) forseventy-two hours. This culture supernatant is termed "conditionedmedium" (C.M.) and was used for purification of NAF. Other media whichwill support ATL-2 cell growth may also be used for culture of thecells.

NAF may be prepared by ultrafiltration of conditioned medium using amembrane having a pore size of 1,000 daltons. Conditioned medium wasfractioned and concentrated 100 fold by ultrafiltration using a YM-2membrane (Amicon, Danvers, Mass.). Components in the conditioned mediumhaving a molecular weight of less than 1,000 daltons pass through themembrane; whereas components of the conditioned medium having amolecular weight greater than 1,000 daltons remain in the concentratedconditioned medium. NAF is retained by the filter and is found in thefraction of conditioned medium containing components having a molecularweight of greater than 1,000 daltons.

NAF may be further purified by filtration of the conditioned mediumfollowed by anion exchange chromatography, preferably using adiethylaminoethyl (DEAE) cellulose with high performance liquidchromatography (HPLC). Active fractions, determined using an in vitroimmune complex kinase technique described in Example 7, or any othersuitable assay that measures tyrosine kinase activity, are pooled,concentrated and subjected to reverse phase chromatography, preferrablyusing a silica matrex column such as c18 (Waters, Inc., Milford, Mass.).Fractions determined to be active through an in vitro immune complexkinase as described in Example 7 or analogous technique have a relativespecific activity of about 1000 times the original ATL-2 conditionedmedium. NAF may be further purified by reverse phase chromatographyusing a second silica matrex column, preferably c18 (referred to hereinas c18#2). Gel electrophoresis using a polyacrylamide gradient gel(Integrated Separation Systems, Hyde Park, Md. or PHorecast System byAmersham, Arlington Heights, Ill.) followed by silver staining of activefractions showed three unique bands at 10 kD, 20 kD and 26 kD which arecharacteristic of NAF.

The proteinaceous substance of the invention may be characterized byphysical properties, its binding specificity for p185, its effects onthe activity of neu protein, and by its effects on p185 bearing cells.NAF stimulates tyrosine kinase activity of p185neu, p185c-neu, andc-erb-B-2. The increase in tyrosine kinase activity may be tested by animmune complex kinase assay, as described in detail in Example 7 herein,or by any other suitable assay that measures tyrosine kinase activity.

The proteinaceous substance of the invention is stable to heat up to atleast 100° C. This stability was determined by treatment of the C.M. for30 minutes at 56° C. and 100° C. The proteinaceous substance exhibitednormal activity after both treatments. In addition, treatment withchymotrypsin and bacterial protease showed that the proteinaceoussubstance of the present invention exhibits protease sensitivity.

NAF activation of p185 tyrosine kinase activity is dependent upon itsspecific binding to p185. Tests using antibodies which are specific forand block distinct epitopes on the extracellular domain of p185indicated that the proteinaceous substance recognizes p185 at particularextracellular domains.

NAF has been found to cause its target protein, p185, to form dimers, oraggregates. This is a common reaction of receptor tyrosine kinases inresponse to their cognate ligands. Both homodimers and heterodimers areformed. Cross-linking studies, described herein, show that NAF causes adose dependent increase in the formation of p185 homodimers in PN-NR6cells. In cells which express both EGF and NAF, such as M1 cells,formation of a heterodimeric receptor structure (p185/EGFR) is dosedependent on treatment with either EGF or NAF.

Some tests show characteristics of NAF which are similar to othertyrosine kinase ligands such as EGF. These tests have indicated that NAFand EGF have similar mechanisms and functions, but are independent ofeach other. In response to exposure to their cognate ligands, receptortyrosine kinases, such as p185, are down-regulated from the cellsurface. NAF causes p-185 to down-regulate. Exposure to NAF caused adecrease in the surface expression of p185 by PN-NR6 cells. Surfaceexpression of EGFR on NE-19 cells was down modulated by the addition ofEGF, but not NAF. This response appears to be temperature sensitivesince incubation with NAF and EGF caused down-regulation of cells whichwere incubated at 37° C. but did not cause down-regulation of cellsincubated at 4° C.

Finally, the effect of NAF on cells which express p185 was examined. NAFwas shown to increase the soft agar growth capability of PN-NR6 cellswhich express p185. NAF had no effect on NE-19 cells which do notexpress p185. Neither NAF or EGF had growth promoting effects on parentNR-6 cells which express neither EGFR nor p185. Other growth factorshave been tested and shown to be unrelated to this growth response.Growth factors such as TGF-α, TGF-β, and PDGF did not cause PN-NR6 orNR6 cells to proliferate.

Although the proteinaceous substance of the invention has initially beenpurified from a human cell line, proteinaceous substances from cells ofother species that have the same or substantially the same bindingaffinity or are capable of binding with p185neu or p185cneu are alsowithin the scope of the present invention. NAF has a molecular weight ofabout 7,000 to about 14,000. daltons based on gel filtrationchromatography and ultrafiltration membrane analysis. NAF may also bepresent in forms having a molecular weight of about 14,000 to about24,000 daltons. Gel electrophoresis using a polyacrylamide gradient gel(Integrated Separation Systems, Hyde Park, MD or PHorecast System byAmersham, Arlington Heights, Ill.) followed by silver staining indicatesthree unique bands at 10 kD, 20 kD and 26 kD in samples of NAF whichhave been purified through a second c18 purification step. The differingmolecular weights of NAF indicate that it may exist in oligomeric, i.e.dimeric or trimeric forms. All the subunits of the proteinaceoussubstance, whether of the smaller or larger molecular weight forms, andmultiples of subunits are within the scope of the invention. Fragmentsor portions of NAF, produced by any method including chemical synthesis,recombinant DNA techniques, degradation or modification of the purifiedproteinaceous substance or any combination of methods, that are capableof binding to p185neu are also within the scope of the invention.Further, fragments or portions of the proteinaceous substance that havebeen chemically modified, or produced in genetically engineered form asa fusion protein or containing amino acid residues not derived from theproteinaceous substance are also within the scope of the invention.

As noted, any combination of the subunits of the dimeric or trimericforms of the proteinaceous substance of invention are also within thescope of the invention. The dimers or trimers may be comprised of two orthree of the same subunits (i.e. homodimers or homotrimers), or two orthree different subunits (i.e. heterodimers or heterotrimers). Thesubunits may be prepared by any suitable method including purificationfrom natural sources, recombinant DNA techniques, chemical synthesis orany combination of methods. Suitable subunits also include fragments orportions of the proteinaceous substance. The subunits may alsooptionally be chemically modified, contain amino acids not derived fromthe proteinaceous substance of the invention, or contain amino acidsequences derived from synthesis as a fusion protein. The ability ofportions or fragments of the proteinaceous substance of the invention,including any other form of the proteinaceous substance of theinvention, to bind to p185 and affect cells expressing p185 on theirsurfaces may conveniently be determined using the assays describedherein in the examples.

The proteinaceous substance of the invention is expected to be useful intreating mammalian diseases and conditions of cells expressing p185 ontheir surfaces. Without wishing to be bound by any particular theory ormode of action, it is presently believed that the proteinaceoussubstance of the invention will modulate the function of cells bybinding to p185 on the surface of the cells which will then alter theenzymatic activity of the cell resulting in an alteration of thefunction of the cell.

For example, secretory epithelial cells express normal cellular p185 ontheir surfaces. Substances bindable with this receptor that stimulatecell growth or metabolism would be useful in situations where cellrepopulation is needed such as after a burn or other type of cellulardestruction or where it is desired to increase or restimulate metabolicproducts of the cell of their effects such as renewed hair growth fromfollicles of the skin.

The proteinaceous substance of the invention will be formulated anddosed according to the specific disorder to be treated, the condition ofthe individual patient, the site of delivery of the proteinaceoussubstance, the method of administration, and other factors known topractitioners. Thus, for the purposes herein, an effective amount of theproteinaceous substance is an amount that is effective to stimulatecellular metabolism, prevent, lessen the worsening of, alleviate or curethe condition for which the proteinaceous substance is administered.

The proteinaceous substance of the invention may be administered to themammal in combination with a pharmaceutically acceptable carrier, suchas sterile water, saline solution or other suffer, or in an emulsion.The proteinaceous substance of the invention may be administered to thecells of the mammal expressing p185 on their surfaces by any convenientroute, such as oral, intravenous, subcutaneous, topical, and other modesof administration. The proteinaceous substance of the invention isadministered to the mammalian patient at a concentration and for alength of time sufficient to modulate the metabolism of the cells. Theparticular concentration given will depend on such factors as thecondition for which it is given, the age and weight of the recipient andthe mode of administration.

The proteinaceous substance may also be combined with cytotoxicsubstances such as radiolabeled molecules and other compounds used fortumor treatment to increase the effect of the proteinaceous substance oncells expressing p185.

For the treatment of tumors overexpressing p185neu on the surfaces ofthe tumor cells or tumors expressing aberrant forms of p185, theproteinaceous substance of the invention, or active fragment or portionthereof, is administered to the tumor or the site of the tumor where itis expected to have an effect on the tumor.

The proteinaceous substance of the invention may also be used in thetreatment of the tumors expressing both p185 and the epidermal growthfactor receptor on the surfaces of the tumor cells. In this embodimentof the invention, the proteinaceous substance, or active fragment orportion thereof, and antibodies specific for the epidermal growth factorreceptor are administered to a mammal having such a tumor in an amounteffective to reduce tumor growth. Methods and reagents for treating suchtumors may be found in copending application Ser. No. 07/386,820 filedJul. 27, 1989 in the name of Mark I. Greene entitled "Methods ofTreating Cancerous Cells With Anti-Receptor Antibodies", the disclosuresof which are hereby specifically incorporated as if fully set forthherein. The proteinaceous substance of the invention is substituted forantibodies specific for p185 and is administered alone or in combinationwith antibodies specific for the epidermal growth factor receptor.

The proteinaceous substance of the invention may be used to diagnosetumors expressing p185 on the surface of the cells such as certainadenocarcinomas, breast, lung, and ovarian cancers, and to identifycells expressing p185. The proteinaceous substance of the invention iscontacted with a tissue sample suspected of containing such tumor cellsunder conditions allowing binding of the proteinaceous substance to thetissue sample. Binding of the proteinaceous substance to cells in thetissue sample which indicates the presence in the tissue sample of tumorcells expressing p185 is then detected using conventional methods andcommercially available reagents, such as antibodies specific for theproteinaceous substance that are labeled with an enzyme, fluorescentmolecule, radiolabel, an electron dense compound such as ferritin, or alight scattering particle such as colloidal gold, or any combinations orpermutations of the foregoing. Antibodies specific for the proteinaceoussubstance may be produced by standard techniques for producingpolyclonal and monoclonal antibodies.

EXAMPLES Abbreviations

p185neu, the product of the rat neu oncogene; p185c-neu, the product ofthe rat neu proto-oncogene; c-erb-B-2, the human homologue of the ratneu gene product; p185 broadly refers to the oncogenic andproto-oncogenic neu gene product from rat, human, and other mammalianand vertebrate homologues of the neu gene product; HTLV-1, human T celllymphotropic virus-1; FCS, fetal calf serum; EGFR, epidermal growthfactor receptor.

The following examples are illustrative, but not limiting of theinvention. Examples 1 and 2 describe the preparation and the physicalcharacteristics of the proteinaceous substance of the present invention.Conditioned medium or partially purified conditioned medium was used inassays performed in Examples 4 through 14. A more purified form of theproteinaceous substance of the invention was used in Examples 15 through21.

EXAMPLE 1 Preparation and Purification of Proteinaceous Substance

ATL-2 cell line is an IL-2-independent HTLV-1 (+) T cell line.Mycoplasm-free ATL-2 cells were maintained in RPMI 1640 mediumcontaining 10% FCS as the culture medium (10% FCS-RPMI 1640) at 37° C.in a humidified atmosphere with 5% CO₂.

For purification of the proteinaceous substance, ATL-2 cells were washedtwice in 1×PBS and cultured at 3×10⁵ /ml in serum-free RPMI 1640 medium/2 mM L-glutamine for seventy-two hours followed by pelleting of thecells. The culture supernatant so produced is termed "conditionedmedium" (C.M.).

C.M. was concentrated 100 fold, from 1 liter to 10 ml, using a YM-2Diaflo membrane (Amicon, Boston, Mass.) with a 1000d cutoff. For use insome assays, concentrated C.M. containing components greater than 1000MW were rediluted to original volume with RPMI medium. Gelelectrophoresis using a polyacrylamide gradient gel (IntegratedSeparation Systems, Hyde Park, MD or PHorecast System by Amersham,Arlington Heights, Ill.) followed by silver staining of some of this twocolumn purified material from the one liter preparation revealed atleast four to five bands of which the 10 kD and 20 kD bands were uniqueto this material. Passed C.M. containing components less than 1000 MWwere used without dilution.

Concentrated C.M. was filter sterilized with a 0.45μ uniflo filter(Schleicher and Schuell, Keene, N.H.) and then further purified byapplication to a DEAE-SW anion exchange column (Waters, Inc. Milford,Mass.) which had been pre-equilibrated with 10 mM Tris-Cl, pH 8.1.Concentrated C.M. proteins representing one liter of original ATL-2conditioned medium per HPLC run were absorbed to the column and theneluted with a linear gradient of 0 mM to 400 mM NaCl at a flow rate of 4ml/min. Fractions were assayed using the in vitro immune complex kinaseassay described in Example 7 with 10% of the appropriate DEAE fraction(1 column purified material) or 1% of the appropriate c18 fractions (twocolumn purified material). The activity which increased the tyrosinekinase activity of p185c-neu in a dose-dependent manner using the invitro immune complex kinase assay described in Example 7 was eluted asone dominant peak across 4 to 5 fractions (36-40) around 220 to 240 mMof NaCl. After HPLC-DEAE purification, the proteins in the activefractions were concentrated and pooled, concentrated and subjected toC18 (silica matrex) reverse phase chromatography (Waters, Inc., Milford,Mass.) (referred to as the c18#1 step or two column purified material).Elution was performed-under a linear gradient of 2-propanol against 0.1%TFA. All the fractions were dialyzed against RPMI 1640 medium to removethe 2-propanol and assayed using the in vitro immune complex kinaseassay, described in Example 7, and a 1% concentration of the appropriatefraction. The activity increasing the tyrosine kinase activity ofp185c-neu was eluted in two peaks. One eluted in fraction 11-13, while asecond, slightly less active peak of activity eluted in fractions 20-23.These two peaks correspond to around 5 to 7% of isopropanol and 11 to14% isopropanol respectively. c18#1 generated fractions 11-13 were usedin the characterization studies. Active fractions obtained from thesecond chromatographic step were pooled, and designated as theproteinaceous substance sample.

A twenty liter preparation employed the same purification strategy. TheDEAE active fractions 35-41 were pooled and subjected to c18chromatography as discussed above. c18#1 fractions 11-13 and 21-24 bothhad dose-dependent activity. The pool of fractions 11-13 was subjectedto an additional c18 chromatographic step (referred to as c18#2 or threecolumn purified material). Again fractions 11-13 and 21-24 had activity.The dose response of fraction 23 as determined by in vitro immunecomplex kinase assay as described in Example 7 may be obtained uponaddition of 0.005% by volume fraction 23 and 0.05% by volume fraction23. This represents the greatest purity achieved.

Molecular weight ranges were determined based on gel filtrationchromatography and ultrafiltration membrane analysis. Near equal amountsof tyrosine kinase activity were retained and passed by a 10,000molecular weight cut off filter. Almost all activity was passed by a30,000 molecular weight cut off filter. Molecular weight ranges foractive chromatographic fractions were determined by comparing fractionscontaining dose-dependent neu-activating activity to the elutionprofiles of a set of protein molecular weight standards (Sigma ChemicalCo., St. Louis, Mo.) generated using the same running conditions. A lowmolecular weight region of activity was identified between 7,000 and14,000 daltons. A second range of activity ranged from about 14,000 toabout 24,000 daltons.

After gel electrophoresis using a polyacrylamide gradient gel(Integrated Separation Systems, Hyde Park, Md. or PHorecast System byAmersham, Arlington Heights, Ill.), silver staining of the three-columnpurified material (c18#2) was done with a commercially available silverstaining kit (Biorad, Rockville Centre, N.Y.). Fraction 21,22,23, and 24from c18#2 purification of the twenty liter preparation were run withmarkers. Fractions 22 and 23 showed the most potent dose response.Several bands unique to these fractions appear at molecular weights 10kD, 20 kD and 26 kD.

EXAMPLE 2 Relative Specific Activity

As shown in Table 1, the relative specific activity of sample increasesan estimated 1000 fold and as much as 3500 fold after purification.

Relative specific activity was determined by comparing the amount ofactivity in the original ATL-2 conditioned medium needed to generate aquantified dose-dependent increase in neu-kinase activity, with theamount of activity in single chromatographic-step purified material, andtwo chromatographic-step purified material needed to generate anequivalent response. Specific activity is the μg of total protein in anNAF-containing sample needed to increase the p185 band intensity, asquantified using a densitometer (LKB Ultrascan, Pharmacia LKBBiotechnology, Piscataway, N.J.), of the in vitro immune complex kinaseassay as described in Example 7, 2.5 fold when compared to untreatedsample. The in vitro immune complex kinase assay is disclosed in Example7. 2.5 fold increases in p185 band intensity were obtained using 10% byvolume original ATL-2 conditioned medium, 1% by volume pooled DEAEactive fractions, and 0.01% c18#1 active fractions. The active fractionsdesignated as c18#1 were used in biochemical and cellularcharacterization studies. The activity of the original ATL-2 conditionedmedium was arbitrarily designated as 7000 units (7 units per ml originalATL-2 supernatant). Values for the total units of activity in thesubsequent DEAE and c18 purification steps were based on the decreasingamount of material (1% by volume for DEAE sample and 0.01% by volume forthe c18#1 material) needed to generate the same increase in bandintensity as does the original ATL-2 supernatant when assayed at 10% byvolume. Using these numbers it was estimated that there are 10 timesmore units and 1000 times more units per ml in the DEAE and c18#1samples respectively when compared to the original ATL-2 conditionedmedium. The increase in relative specific activity of approximately fourfold for the DEAE generated material and approximately 250 fold for thec18#1 generated material are consistent with resolving capabilities ofthese two chromatographic techniques.

Along with the increase in relative specific activity, two otherindicators of the increased purity may be noted. First, the activity ofthe pooled c18#1 separation was far greater than the originalsupernatant. Dimerization, down-modulation, and proliferationexperiments were cleaner and more readily reproducible with c18#1material, indicating an increased purity. The purified molecules wereactive even at reduced temperatures (<27° C.) in terms of dose responsestudies. The increased activity and function at lower temperatures alsoreflect increased proteinaceous substance purity.

                  TABLE 1                                                         ______________________________________                                        Specific Activity of Proteinaceous Substance                                  at Different Stages of Purity                                                                                Activity                                              Concen-         Total   (total                                                tration                                                                              Volume   Protein unit)  Relative                                       (μg/ml)                                                                           (ml)     (μg) (unit/μg)                                                                         Activity                                ______________________________________                                        Culture  7        1000     7000  7000   1                                     Supernatant                                                                   DEAE     17       40       680   2800   4.11                                  Frac. 36-40)                                                                  C18 No. 1                                                                              0.63     3        1.89  2100   1111                                  (Frac. 11-13)                                                                 ______________________________________                                    

EXAMPLE 3 Maintenance and Characterization of Cell Lines

All cell lines were maintained in RPMI 1640 medium containing 10 % FCSand 2mM L-glutamine at 37° C. in a humidified atmosphere with 5 % CO₂.NR6 cells are a subclone of Swiss 3T3 cells which do not expressdetectable levels of EGFR, do not respond to EGF and do not express anyp185. NR6 cells were co-transfected with PSV2-NEO and PSV2-neuN by theCa⁺⁺ phosphate precipitation technique. The resulting cell line, calledPN-NR6 (proto-oncogenic neu), expresses p185c-neu at high levels. NE-19cells are NR6 cells transfected with a human EGFR gene and express EGFRat high levels. The M1 cell line was derived from NR6 cells andexpresses EGFR and p185c-neu at high levels. These cell lines weredescribed by Kokai et al., (1989) Cell 58:287. A431 cells (American TypeCulture Collection) are a human spidermold carcinoma-derived cell linewhich express high levels of EGFR. SKBR111 cells are a human breastadenocarinoma-derived cell line which expresses both EGFR and c-erbB-2.B104 cells were derived from an ethyl-nitrosouria-induced ratneuroectodermal tumor. B104-1'-1'cells are NIH/w cells transfected withDNA originally isolated from the B104 cell line and later found toencode the neu oncogene. The NIH/w cell line is an NIH3T3 cell subclonewhich displays a low frequency of spontaneous tumor formation and whichlacks EGFR.

EXAMPLE 4 Anchorage-Independent Growth Assay

Anchorage-independent growth capability was determined by assessing thecolony-forming efficiency of cells suspended in soft agar. Allexperiments to determine colony-forming efficiency were conducted using60 mm tissue culture dishes containing a 3 ml cell free feeder layer anda 1 ml top layer in which the cells were suspended. Feeder layersconsisted of 0.24% agarose RPMI-1640 supplemented with 10% fetal calfserum, 2 mM L-glutamine. Overlayers contained 1×10⁴ cells in 0.18%agarose RPMI-1640 supplemented with 10% fetal calf serum, 2 mML-glutamine. When sample containing proteinaceous substance was added tosoft-agar cultures it was incorporated into the top layer only. Colonieslarger than 0.5 mm in diameter were counted using a dissectingmicroscope at seven days. 4 random field (×100) were chosen from each 6cm dish. Mean number was determined from these four fields to representeach 6 cm dish. Each experimental group represents the mean oftriplicate dishes. In tables, a parenthesis shows the standard deviationof the triplicate samples.

EXAMPLE 5 Anchorage-Independent Growth Assay--Heat Treated ConditionedMedium

Conditioned medium (C.M.) were treated at 56° C. or 100° C. for thirtyminutes. As shown in Table 2, both non-treated and heat-treated C.M.increased the colony formation of B104-1'-1'cells. This activity wasstable even after heat-treatment.

                  TABLE 2                                                         ______________________________________                                        Anchorage Independence Assay B104-1'-1' Cells                                 Heat Treatment of Conditioned Medium                                                   Concentration of Conditioned Medium                                           0%       1.0%      10%                                               ______________________________________                                        No Treatment          10.00 (0.81)                                                                            20.00 (5.88)                                  56° C. treatment                                                                              7.50 (0.81)                                                                            19.75 (6.13)                                  100° C. treatment                                                                            14.00 (5.59)                                                                            18.75 (2.98)                                  Control    5.25 (2.62)                                                        ______________________________________                                    

EXAMPLE 6 Anchorage-Independent Growth Capability of FractionatedConditioned Medium

The ability to increase the colony formation of B104-1'-1' was containedin the C.M. concentrated by YM-2 membrane, which contains the componentshaving a molecular weight of greater than 1000 dalton.Anchorage-independent growth capability of fractionated conditionedmedium is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Anchorage-Independent Growth Capability of                                    Fractionated Conditioned Medium                                                         Concentration of C.M.                                                         0%       1.0%      10%                                              ______________________________________                                        No Treatment           17.50 (6.45)                                                                            29.25 (7.13)                                 Concentrated           19.25 (5.61)                                                                            56.25 (8.18)                                 Passed                 15.25 (4.19)                                                                            19.00 (5.29)                                 Control     14.00 (4.19)                                                      ______________________________________                                    

EXAMPLE 7 Immune Complex Kinase Assay

This assay reflects the differences in the autophosphorylation activityof immunoprecipitated p185 driven by pre-incubation of PN-NR6 celllysate with varying amounts of ATL-2 conditioned medium (C.M.) orproteinaceous substance and is referred to hereinafter as ned-activatingactivity. The cell lines are described in greater detail in Example 3.

Cell lines used in the immune complex kinase assay were obtained,prepared and cultured according to the methods disclosed in Kokai etal.,Cell 58: 287-292, (Jul. 28, 1989) the disclosures of which are herebyincorporated by reference as if fully set forth herein, and U.S.application Ser. No. 386,820 filed Jul. 27, 1989 in the name of Mark I.Greene entitled "Methods of Treating Cancerous Cells With Anti-ReceptorAntibodies", the disclosures of which are hereby incorporated byreference as if fully set forth herein.

Cell lines were all maintained in DMEM medium containing 5% FCS as theculture medium (5% FCS-DMEM) at 37° C. in a humidified atmosphere with5% CO₂.

Dense cultures of cells in 150 mm dishes were washed twice with coldPBS, scraped into 10 ml of freeze-thaw buffer (150 mM NaCl, 1 mM MgCl₂,20 mM Hepes, pH 7.2, 10% Glycerol, 1 mM EGTA, 1% Aprotinin), andcentrifuged (600×6, 10 minutes). Cell pellets were resuspended in 1 mlLysis buffer (50mM Hepes, PH 7.5, 150 mM NaCl, 3% Brij 35, 1 mM EDTA,1.5 mM MgCl₂, 1% Aprotinin, 1 mM EGTA, 30 μM Na₃ VO₄, 10% Glycerol)rotated for thirty minutes at 4° C. All chemicals were from SigmaChemical Co., St. Louis, Mo., unless otherwise indicated. The insolublematerials were removed by centrifugation at 40,000×g for thirty minutes.The clear supernatant which was subsequently used is designated as celllysate.

The cell lysates were incubated for fifteen minutes with 50 μl of 50%(volume/volume) Protein A-sepharose (Sigma Chemical Co., St. Louis,Mo.), and centrifuged for two minutes to preclear the lysates. 50 μlaliquots of precleared cell lysate were incubated on ice for fifteenminutes with conditioned medium, proteinaceous substance, or otherfactors as specified, in a final volume of 1 ml with lysis buffer. Thesample was then incubated with 5 μg of 7.16.4. monoclonal antibody,which recognizes the extracellular domain of the p185neu and p185 c-neu,or other appropriate antibodies, for twenty minutes on ice, followed bya twenty minute incubation with 50 μl of 50% (vol/vol) proteinA-sepharose with rotation at 4° C. Immune complexes were collected bycentrifugation, washed four times with 500 μl of washing buffer (50mMHepes, pH 7.5, 0.1%, Brij 35, 150 mM NaCl, 2 mM EDTA, 1% Aprontinin, 30μM Na₃ VO₄), then twice with reaction buffer (20 mM Hepes (pH7.4), 3 mMMnCl₂ and 0.1% Brij 35, 30 μM Na₃ VO₄). Pellets were resuspended in 50μl of reaction buffer and [Gamma- ³² P]-ATP (Amersham, ArlingtonHeights, Ill.) was added giving a final concentration of 0.2 μM. Thesamples were incubated at 27° C. for twenty minutes or at 4° C. for 25minutes with purer samples. The reactions were terminated by addition of3×SDS sample buffer containing 2 mM ATP and 2 mM EDTA and thenincubating them at 100° C. for five minutes. The samples were thensubjected to SDS-PAGE analysis on 10% acrylamide gels. Gels werestained, dried, and exposed to Kodak XAR or XRP film with intensifyingscreens.

EXAMPLE 8 Effect of C.M. on Kinase Activity of PN-NR6 and M1 Cells

Lysates of PN-NR6 and M1 cells were treated as described in Example 7and received the following: No addition (control); 0.1% of C.M.; 1.0% ofC.M.; or 10% of C.M. The addition of conditioned medium increasedtyrosine kinase activity of p185c-neu from PN-NR6 and M1 cells at allconcentrations of conditioned medium. There was a two to four foldincrease in p185 band intensity in lanes representing 10% conditionedmedium treatment as compared to lanes with no conditioned mediumtreatment.

EXAMPLE 9 Effect of Conditioned Medium On Tyrosine Kinase Activity OfEpidermal Growth Factor Receptor

Tyrosine kinase activity was determined using the immune complex kinaseassay described in Example 7. Lysates of A431 cells were treated asdescribed and received the following: No addition (control), 0.1 ng/ml1.0 of EGF; 1.0 ng/ml of EGF; 10 ng/ml of EGF; 0.1% of C.M.; 1.0% ofC.M.; or 10% of C.M. The addition of EGF increased the tyrosine kinaseactivity of EGFR from A431 cells. The addition of conditioned medium didnot increase the tyrosine kinase activity of EGFR from A431 cells.Tyrosine kinase activity of epidermal growth factor receptor (EGFR) isincreased by addition of EGF but not conditioned medium. This indicatesthe receptor specificity of the factor.

EXAMPLE 10 Effect Of Conditioned Medium On Phosphorylation Of Histone ByKinase Of p185c-neu

Tyrosine kinase activity was determined using the immune complex kinaseassay described in Example 7. Lysates of PN-NR6 cells were treated asdescribed and received the following: No addition; 1.0% of C.M.; or 10%of C.M. 2μl of histone (2 mg/ml) was added with ³² P-r-ATP as substratesof tyrosine kinase of p185c-neu. The phosphorylation of histones bytyrosine kinase of p185c-neu was increased two to six fold by additionof conditioned medium.

EXAMPLE 11 Effect Of Heat Treatment On Tyrosine Kinase Activity Ofp185c-neu

Tyrosine kinase activity was determined using the immune complex kinaseassay described in Example 7. C.M. were treated at 56° C. or 100° C. forthirty minutes. Lysates of PN-NR6 cells were then treated as describedand received the following: No addition; 1.0% of heat-treated ornon-treated C.M.; 10% of heat-treated or non-treated C.M. The activitycontained in the C.M. was stable after heat treatment at either 56° C.or 100° C. for thirty minutes. Thus the p185c-neu tyrosine kinaseactivating activity in the C.M. is not affected by heat treatment.

EXAMPLE 12 Tyrosine Kinase Activity Of Fractionated Conditioned Medium

Conditioned medium was concentrated as described in Example 1.Concentrated C.M. containing components greater than 1000 MW wererediluted to original volume by distilled water and used in assays.Passed C.M. containing components less than 1000 MW were used withoutdilution.

Tyrosine kinase activity was determined using the immune complex kinaseassay described in Example 7. Lysates of PN-NR6 cells were treated asdescribed and received the following: No addition; 1.0% of C.M.; 10% ofC.M.; non-treated C.M.; concentrated C.M.; passed C.M. Increasedtyrosine kinase of p185c-neu was contained in concentrated C.M. with amolecular weight greater than 1000. Near equal amounts of activity wereretained and passed by a 10,000 molecular weight cut off filter. Almostall activity was passed by a 30,000 molecular weight cut off filter.

EXAMPLE 13 Effect Of Protease On Tyrosine Kinase Activity Of ConditionedMedium

Tyrosine kinase activity was determined using the immune complex kinaseassay described in Example 7. A first portion of C.M. was treated with100μg alpha-chymotrypsin (Sigma, St Louis, Mo.) for two hours at 37° C.The reaction was stopped by adding 2-fold molar excess ofchymotrypsin-inhibitor (Sigma, St. Louis, Mo.) A second portion of C.M.was treated with 100 μg bacterial protease (Sigma, St. Louis, Mo.) fortwo hours at 37° C. The reaction was stopped by heat-inactivating theprotease by boiling for five minutes. Lysates of PN-NR6 cells weretreated as described and received the following: A) No addition; B) C.M.treated by chymotrypsin, no inhibitor added; C) C.M. treated bychymotrypsin-inhibitor only; D) C.M. treated by chymotrypsin, thentreated by inhibitor; E) C.M. treated by chymotrypsin which waspreviously inactivated by chymotrypsin inhibitor; F) C.M. treated bybacterial protease, then heat-inactivated; G) C.M. treated by bacterialprotease which was pre-inactivated by heat treatment; and H) non-treatedC.M.

After SDS/PAGE, the band intensity of protease treated p185c-neu sampleswas less than those samples that were not treated with protease. Bandintensity of protease treated samples was also less intense than bandsof samples treated with protease wholly or partly inactivated withprotease inhibitor or heat treatment. These data indicate that thep185c-neu-specific activity in the C.M. is sensitive to digestion bychymotrypsin and bacterial protease.

EXAMPLE 14 Effect of Conditioned Medium On Binding Activity Of AntibodyTo p185-neu

Tyrosine kinase activity was determined using the immune complex kinaseassay described in Example 7. C.M. was added before immunoprecipitationby 7.16.4 monoclonal antibody. Monoclonal antibody 7.16.4 recognizes theextracellular domain of p185neu and p185c-neu. p185c-neu (from PN-NR6cells) was immunoprecipitated by 7.16.4 monoclonal antibody and immunecomplexes were washed by washing buffer once and then immune complexeswere incubated with C.M. for fifteen minutes at 4° C. After these steps,the immune complex kinase assay, set forth in Example 7, was performedas described herein. Lysates of PN-NR6 were treated as described andreceived the following: No addition; 1.0% of C.M.; and 10% of C.M.Tyrosine kinase activity of C.M. was blocked by pretreatment with 7.16.4monoclonal antibody. These data show that a component in the conditionedmedium binds with p185c-neu.

EXAMPLE 15 Effect of ATL-2 secreted growth modulating substances on thekinase activity of p185c-neu

Growth modulating substances such as ADF (IL-2 receptor inducingfactor), TGF-α (transforming growth factor α), TGF-β (transforminggrowth factor β), PDGF (platelet derived growth factor), IL-1(interleukin 1) and IL-6 (interleukin 6) which are secreted from theATL-2 cell line were tested using the in vitro immune complex kinaseassay, as set forth in Example 7. None of these factors-affected thekinase activity of p185c-neu. Representative of results obtained wastreatment of PN-NR6 lysate with 10 or 100 units of IL-1 or 25 or 250units of IL-6 which resulted in no increase in kinase activity.

EXAMPLE 16 Effect of NAF and EGF on the phosphotyrosine content of p185and EGFR expressed in identical fibroblast cell background

Western blot analysis as described in Wada et al., (1990) Cell 61:1339,was employed to demonstrate that the quantity of phosphotyrosine in p185was specifically increased by the addition of NAF. Intact PN-NR6(p185+/EGRF-) and NE-19 (EGRF+/p185-) cells (2×10⁶) in 10 cm culturedishes were incubated for 8 minutes at 37° C. with 0, 0.1 or 1.0% byvolume NAF or0, 10 or 100 ng/ml EGF. The cells were washed three timeswith cold phosphate buffered saline containing 400 EDTA, 10 mM sodiumpyrophosphate, 10 mM sodium fluoride and 400 μM sodium orthovanadate andwere lysed in PI/RIPA buffer (1% Triton X-100, 1% Deoxycholate, 0.1%SDS, 0.15 M NaCl , 0.01 M sodium pyrophosphate pH 7.4,1% trasylol, 1 μMPMSF, 2 mM EBTA, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 400μM sodium orthovanadate, 10 mM iodo-acetamide, and 1 mM ATP) for 30minutes. Pre-cleared supernatants were subjected to immunoprecipitationwith monoclonal antibody 7.16.4 (which reacts specifically with ratp185) or anti-EGFR carboxy terminal antibodies (provided by Dr. StuartDecker, Rockefeller University, New York, N.Y.) respectively.Immunoprecipitates were washed 2 times with washing buffer (0.1% TritonX-100, 0.4 mM EDTA, 10 μM sodium fluoride, 10mM Sodium pyrophosphate,400 μM Sodium orthovanadate, 0.01 M sodium phosphate, pH 7.4). Washedimmunoprecipitates were boiled in SDS-sample buffer (pH6.8) (3% SDS, 10%glycerol, 5% 2ME, 0.4% bromophenol blue) and analyzed on 10% SDS-PAGEgels. Proteins were transferred to nitrocellulose and detected byanti-phosphotyrosine antibody PY-20 MAb (ICN Biomedicals, Inc., CostaMesa, Calif.) as described in Wada et al., (1990) Cell 61:1339.

The amount of phosphotyrosine in p185c-neu from PN-NR6 cells(p185+/EGFR-) was increased by addition of 0.1% or 1.0% "NAF in adose-dependent manner while addition of 10 and 100 ng/ml EGF had noeffect under the same conditions. The amount of phosphotyrosinedetectable in EGFR from NE-19 cells (EGFR+/p185-) was increased byaddition of 10 or 100 ng/ml EGF but was not increased by addition of0.1% or 1.0% NAF. NAF affected the tyrosine kinase activity of p185c-neuexpressed in the NR6 cell background (PN-NR6 cells) yet had no effect onEGFR in the identical NR6 cell background (NE-19 cells) again indicatingthat the observed effects are not mediated by EGF and its receptor.Controls with no addition of NAF or EGF produced no response in eithercell line.

EXAMPLE 17 NAF Interacts with the Extracellular Domain of the neuProtein p185

Monoclonal antibodies specific for distinct epitopes on theextracellular domain of p185 were examined for their ability to blockthe NAF-mediated increase of p185 kinase activity using the immunecomplex kinase assay as described in Example 7. PN-NR6 cells werepre-incubated with 1 μg/ml antibody or no antibody (control) at 4° C.for 30 minutes, and then washed twice with cold PBS. Cells were thenexposed to 1% (by volume) NAF or no NAF (control) and incubated at 37°C. for 10 minutes. Cells were lysed, p185 was immunoprecipitated with7.16.4, an anti-p185 antibody, and the immune complex kinase assay wasperformed as described in Example 7.

Pre-incubation of PN-NR6 cells (p185+/EGFR-) with purified p185extracellular domain specific monoclonal antibody 7.16.4 (Drebin et al.,(1984) Nature 312:545; Drebin et al., (1985) Cell 41:695) blocked theNAF-induced activation of p185, while an irrelevant, isotype matchedmonoclonal antibody 9BG5, an IgG2a monoclonal antibody reactive with thereovirus type 3 hemaglutanin (Drebin et al., (1988) Oncogene 2:273) didnot block the NAF-induced activation of p185. Monoclonal antibody 7.9.5recognizes a distinct extracellular domain of p185 (Drebin et al.,(1988) Oncogene 2:273) and only partially blocked the NAF-inducedactivation of p185. Similarly, pre-incubation of cells with suramin,which has been shown to block EGF and PDGF from binding to theirreceptors (Yarden and Weinberg,(1989) Proc. Natl. Acad. Sci. U.S.A.86:3179 and L. T. Williams, et al., (1984) J. Biol. Chem. 259:5287)blocked the NAF-induced activation of p185. In addition, western blotanalysis with anti-phosphotyrosine monoclonal antibodies was performedusing a mutant p185, termed p185-D4 which contains a 523 based pairdeletion corresponding to amino acids 475 through 648 (in frame) of thesecond cysteine rich domain of the extracellular region of p185c-neu.NAF did not increase the kinase activity of this p185 mutant(transfected into NR6 cells) that lacks this portion of theextracellular putative ligand-binding domain of p185c-neu. Collectivelythese results indicate that NAF interacts with discrete parts of the neuprotein extracellular domain.

EXAMPLE 18 Effect of NAF on p185 Dimerization

Receptor tyrosine kinases are induced by their cognate ligands to formreceptor aggregates. Homodimeric and heterodimeric species of p185-neuhave previously been described. The point mutation (from adenine toglutamic acid at residue 664) in the transmembrane region of p185-neu isable to facilitate homodimer formation (Weiner, et al., (1989) Nature pb338:230).

Cross-linking studies were performed to determine the effect NAF on p185dimerization. Chemical cross linking was done as described by Wada etal., (1990) Cell 61:1339. 3×10⁶ cells in 10 cm culture dishes wereplated and incubated with 5% FCS overnight. Medium was then replaced byITS-DMEM and cultured overnight. Cells were incubated with or withoutITS-DMEM supplemented with or without the indicated amount of NAF or EGFat 37° C. for 8 minutes and them washed twice with cold PBS. Five ml ofPBS containing 4 mM BS₃ (Pierce, Rockford, Ill.) was added and cellswere incubated at 22° C. for 30 minutes. The cross linking reaction wasquenched by addition of quenching buffer. Cells were then solubilizedand their lysates were subjected to immunoprecipitation with 7.16.4antibody. Proteins were then separated on 4%-7.5% gradient SDS-PAGEminigels and transferred to nitrocellulose. Monomeric and dimeric formsof p185c-neu were detected with anti-phosphotyrosine MAb PY-20 (ICNBiomedicals, Inc., Costa Mesa, Calif.) and DBW 2 (anti-p185intracellular domain) antibodies. Antibodies were detected with ¹²⁵I-Protein A (New England Nuclear, Boston, Mass.).

Cross-linking studies revealed that the amount of p185c-neu homodimersin PN-NR6 cells (p185+/EGFR-) was increased in a dose dependent mannerwith exposure to 0.1% or 1.0% NAF. A control with no addition of NAFproduced no response. The amount of phosphotyrosine in the p185c-neuhomodimers also increased in a dose-dependent manner with addition of0.1% or 1.0% NAF. Heterodimeric species of p185c-neu have recently beendescribed Kokai et al., (1989) Cell 58:287; Wada et al., (1990) Cell61:1339. In M1 cells, which express high levels of both p185c-neu andEGFR, Kokai, et al., (1989) Cell 58:287, p185c-neu/EGFR heterodimersexist and EGF treatment of M1 cells also increased the amount ofdetectable p185c-neu/EGFR heterodimers in a dose-dependent manner.

EXAMPLE 19 Effect of NAF and EGF on p185 and EGFR Internalization

In response to exposure to their cognate ligands, receptor tyrosinekinases are down regulated from the cell surface. The ability of NAF todown modulate p185c-neu from the surface of PN-NR6 cells was examined byquantitative immunological assessment of the amount of cell surface p185on PN-NR6 cells after treatment with NAF.

p185c-neu down modulation assay was performed as described by Yarden andWeinberg,(1989) Proc. Natl. Acad. Sci. U.S.A. 86:3179. Cells werecultured overnight in DMEM containing ITS and incubated with NAF or EGFfor the indicated time period. 1×10⁵ PN-NR6 or NE19 cells in 24 welldishes (Costar, Cambridge, Mass.) were seeded and incubated overnight inDMEM medium containing 5% fetal calf serum (FCS). Those cells werewashed with DMEM and incubated 1 hour with DMEM and then incubated withbinding buffer [DMEM supplemented with 20mM Hepes (pH 7.2) and 0.1%bovine serum albumin] with or without 1% NAF(by volume) or 50 ng/ml EGF.After incubation at 37° C., the monolayer was washed with DMEM andincubated at 4° C. with 2 μg/ml of the 7.16.4 or anti-EGFR antibody.This monoclonal antibody recognizes the extracellular domain of humanEGF receptor and was provided by Dr. M. Herlyn of the Wistar Institute,Philadelphia, Pa. After a 2 hour incubation, cell-bound antibody wasdetermined by a 45-minute incubation with ¹²⁵ I-labeled protein A.

Surface expression of p185c-neu on PN-NR6 cells was decreased by 30% at30 minutes and by 40% at 90 minutes after the addition of NAF while EGFhas no effect on the internalization of p185c-neu in these same cells.Surface EGFR on NE-19 cells was down-modulated by the addition EGF, butwas not down-modulated by proteinaceous substance. When the cells wereincubated with NAF or EGF at 4° C. rather than 37° C., neither p185c-neunor EGFR could be down modulated on either cell type.

EXAMPLE 20 Effect of NAF and Other Factors on Cell Growth measured by [³H]-Thymidine Incorporation

The effect of NAF on the growth of cells was assessed using tritiatedthymidine incorporation. Subconfluent cells were trypsinized and 1×10⁴cells were suspended in DMEM medium containing 10% FCS and placed in96-well plate. Following cell attachment overnight, the medium wasreplaced with DMEM medium suplemented with ITS (a defined culture mediumhaving insulin, transferrin and selenium as three of its most commoncomponents (Collaborative Research, Bedford, Mass.). Incubation wascontinued in this serum-free medium for an additional 48 hours.Thereafter, cells were exposed to NAF, EGF (epidermal growth factor),TGF-α (transforming growth factor e), TGF-β (transforming growth factorβ), or PDGF (platelet derived growth factor) factors for 16 hours. Cellsreceived a 6 hour pulse of [³ H] thymidine (0.5 uCi/ml) prior toharvest.

NAF increased the relative levels of DNA synthesis in cultures of PN-NR6cells (p185+/EGFR). 1.0% NAF increased the DNA synthesis three fold. NAFdid not affect the levels of DNA synthesis in cultures of NE-19 cells(p185-/EGFR+). DNA synthesis in cultures of NE-19 cells was increasedtwo fold by the increase of EGF from 10 ng/ml to 100 ng/ml. Neither EGF,TGF-α (10 ng/ml or 100 ng/ml), TGF-β (10ng/ml), or PDGF (10 ng/ml)factors significantly affected the DNA synthesis cultures of PN-NR6cells.

EXAMPLE 21 Anchorage-Independent Growth Assays--Capability of Factors

Anchorage-independent growth assays were performed as described inExample 4 to determine the growth capabilities of NAF, EGF, TGF-α,TGF-β, and PDGF on PN-NR6, NE-19 and NR-6 cell lines. Results are setforth in Table 4.

PN-NR6 cells do not oridinarily form colonies in soft agar while NIH/3T3transfectants which overexpress EGFR form colonies in soft agar onlyupon addition of EGF (DiFiore et al. (1987) Cell 51:1063). NAF alsoincreased the soft agar growth capability of PN-NR6 cells (p185+/EGFR-),but had no effect on NE-19 cells (EGFR+/p185-). Conversely, EGFincreased the soft-agar growth capability of NE-19 cells, but had noeffect on the neu-bearing PN-NR6 cells. The parent NR-6 cells(p185-/EGFR-) were not affected by EGF or by NAF. Thus, the growthpromoting effects of the proteinaceous substance were seen only in cellsthat express p185c-neu. Furthermore it is unlikely that other factorsare responsible for these results since TGF-α, TGF-β, and PDGF did notcause PN-NR6 or NR6 cells to proliferate or form colonies in soft agar.

                  TABLE 4                                                         ______________________________________                                        Effect of Growth Factors on Cell Growth                                       Factor   Concentration                                                                             PN-NR-6   NE-19 NR-6                                     ______________________________________                                        --       --          0         0     0                                        NAF      0.1         6         0     0                                        (%)      1.0         25        0     0                                        EGF      10          0         22    0                                        (ng/ml)  100         0         30    0                                        TGF-α                                                                            10          0          n.d* n.d                                      (ng/ml)  100         0         n.d   n.d                                      TGF-β                                                                             1           0         n.d   n.d                                      (ng/ml)  10          0         n.d   n.d                                      PDGF     1           0         n.d   n.d                                      (ng/ml)  10          0         n.d   n.d                                      ______________________________________                                         *n.d = not determined                                                    

We claim:
 1. A purified proteinaceous substance which is bindable withp185, wherein said proteinaceous substance:increases the activity of thetyrosine kinase contained in p185 but does not increase the activity oftyrosine kinase of epidermal growth factor receptor; induces p185dimerization; induces p185 internalization; affects the growth of cellswhich express p185 in a dose dependent manner; is heat stable from about56° C. to about 100° C.; is degradable by protease; and has a molecularweight of from about 7,000 to about 14,000 daltonsor a fragment thereofhaving binding affinity for p185.
 2. A dimer of the proteinaceoussubstance of claim
 1. 3. A trimer of the proteinaceous substance ofclaim
 1. 4. A method of screening for a mammalian tumor that has p185 onthe surfaces of the tumor cells comprising the steps of:contactingsuspected tumor cells with the proteinaceous substance of claim 1 underbinding conditions; and detecting bound proteinaceous substance, whereinthe presence said bound proteinaceous substance indicates the presenceof p185.
 5. The method of claim 4 wherein said proteinaceous substancea)is a fragment of a proteinaceous substance that:i) increases theactivity of the tyrosine kinase contained in p185 but does not increasethe activity of tyrosine kinase of epidermal growth factor receptor; ii)is heat stable from about 56 to about 100° C.; iii) is degradable byprotease; and iv) has a molecular weight of from about 7,000 to about14,000 daltons; and b) has binding affinity for p185.
 6. The purifiedproteinaceous substance of claim 1 wherein said proteinaceoussubstancea) is a fragment of a proteinaceous substance that:i) increasesthe activity of the tyrosine kinase contained in p185 but does notincrease the activity of tyrosine kinase of epidermal growth factorreceptor; ii) is heat stable from about 56° to about 100° C.; iii) isdegradable by protease; and iv) has a molecular weight of from about7,000 to about 14,000 daltons; and b) has binding affinity for p185. 7.A purified proteinaceous substance which is bindable with p185, whereinsaid proteinaceous substance:increases the activity of the tyrosinekinase contained in p185 but does not increase the activity of tyrosinekinase of epidermal growth factor receptor; is heat stable from about56° to about 100° C.; is degradable by protease; and has a molecularweight of from about 7,000 to about 14,000 daltons.
 8. A dimer of theproteinaceous substance of claim
 7. 9. A trimer of the proteinaceoussubstance of claim
 7. 10. A method of screening for a mammalian tumorthat has p185 on the surfaces of the tumor cells comprising the stepsof:contacting suspected tumor cells with the proteinaceous substance ofclaim 7 under binding conditions; and detecting bound proteinaceoussubstance, wherein the presence said bound proteinaceous substanceindicates the presence of p185.
 11. An isolated protein fraction fromATL-2 cells comprising a purified proteinaceous substance which isbindable with p185, wherein said proteinaceous substance:increases theactivity of the tyrosine kinase contained in p185 but does not increasethe activity of tyrosine kinase of epidermal growth factor receptor; isheat stable from about 56° to about 100° C.; is degradable by protease;and has a molecular weight of from about 7,000 to about 14,000 daltons.12. A dimer of the proteinaceous substance of claim
 11. 13. A trimer ofthe proteinaceous substance of claim
 11. 14. A method of screening for amammalian tumor that has p185 on the surfaces of the tumor cellscomprising the steps of:contacting suspected tumor cells with theisolated protein fraction of claim 11 under binding conditions; anddetecting bound proteinaceous substance, wherein the presence said boundproteinaceous substance indicates the presence of p185.